Transfection complexes

ABSTRACT

The invention provides a peptide having at least 3 amino acids comprising an amino acid sequence selected from a) X 1 SM [SEQ.ID.NO.:1] b) LX 2 HK [SEQ.ID.NO.:2] c) PSGX 3 ARA [SEQ.ID.NO.:9] d) SX 4 RSMNF [SEQ.ID. NO:16] e) LX 5 HKSMP [SEQ.ID.NO.:18] in which X is a basic amino acid residue, X 1  is Q or P, X 2  is A or T, X 3  is an acidic amino acid residue and X 4  is P or Q, the invention further provides non-viral cell-targeting vector complexes and methods associated therewith.

[0001] The present invention relates to peptides for use in an improvedmethod of transfecting cells.

[0002] The term “transfection” is used herein to denote the introductionof a nucleic acid into a cell. The nucleic acid may be of any origin,and the recipient cell may be prokaryotic or eukaryotic.

[0003] Gene therapy and gene vaccination are techniques that offerinteresting possibilities for the treatment and/or prophylaxis of avariety of conditions, as does anti-sense therapy. Such techniquesrequire the introduction of a DNA of interest into target cells. Theability to transfer sufficient DNA to specific target cells remains oneof the main limitations to the development of gene therapy, anti-sensetherapy and gene vaccination. Both viral and non-viral DNA deliverysystems have been proposed. In some cases RNA is used instead of DNA.

[0004] Receptor-mediated gene delivery is a non-viral method of genetransfer that exploits the physiological cellular process,receptor-mediated endocytosis to internalise DNA. Examples includevectors targeted against insulin receptors, see for example, Rosenkranzet al Experimental Cell Research 199, 323-329 (1992), asialoglycoproteinreceptors, see for example, Wu & Wu, Journal of Biological Chemistry262, 4429-4432 (1987), Chowdhury et al Journal of Biological Chemistry268, 11265-11271 (1993), and transferrin receptors, see for example,Ciriel et al, Proc. Natl. Acad. Sci. USA 88, 8850-8854 (1991). Furtherexamples of vectors include monoclonal antibodies targeting receptors onneuroblastoma cells (Yano et al, 2000), folate conjugated to liposomes(Reddy & Low 2000, Reddy et al. 1999), galactose for targeting livercells (Han et al. 1999 Bettinger et al. 1999) and asialogylcoprotein,also for liver cells (Wu et al. 1991).

[0005] Receptor-mediated non-viral vectors have several advantages overviral vectors. In particular, they lack pathogenicity; they allowtargeted gene delivery to specific cell types and they are notrestricted in the size of nucleic acid molecules that can be packaged.Gene expression is achieved only if the nucleic acid component of thecomplex is released intact from the endosome to the cytoplasm and thencrosses the nuclear membrane to access the nuclear transcriptionmachinery. However, transfection efficiency is generally poor relativeto viral vectors owing to endosomal degradation of the nucleic acidcomponent, failure of the nucleic acid to enter the nucleus and theexclusion of aggregates larger than about 150 nm from clathrin coatedvesicles.

[0006] Desirable properties of targeting ligands for vectors are thatthey should bind to cell-surface receptors with high affinity andspecificity and mediate efficient vector internalisation. Short peptideshave particular advantages as targeting ligands since they arestraightforward to synthesise in high purity and, importantly for invivo use, they have low immunogenic potential.

[0007] WO 98/54347 discloses a mixture comprising an integrin-bindingcomponent, a polycationic nucleic acid-binding component, and a lipidcomponent, and also discloses a complex comprising

[0008] (i) a nucleic acid, especially a nucleic acid encoding a sequenceof interest,

[0009] (ii) an integrin-binding component,

[0010] (iii) a polycationic nucleic acid-binding component, and

[0011] (iv) a lipid component.

[0012] The complex is primarily an integrin-mediated transfectionvector.

[0013] Integrins are a super-family of heterodimeric membrane proteinsconsisting of several different α and β subunits. They are important forattachment of cells to the extracellular matrix, cell-cell interactionsand signal transduction. Integrin-mediated internalisation proceeds by aphagocytic-like process allowing the internalisation of bacterial cellsone to two micrometers in diameter (Isberg, 1991). Targeting ofnon-viral vectors to integrins, therefore, has the potential totransfect cells in a process that mimics infection of cells by pathogensand avoids the size limitation imposed by clathrin-coated vesicles inreceptor-mediated endocytosis.

[0014] It is considered that the components described in WO 98/54347associate electrostatically to form the vector complex, the vector beingof the lipopolyplex type. The vector complexes of WO 98/54347 are foundto transfect a range of cell lines and primary cell cultures with highefficiency, with integrin specificity and with low toxicity. Forexample, vascular smooth muscle cells are transfected with 50%efficiency, endothelial cells with 30% efficiency and haematopoieticcells with 10% efficiency. Furthermore, in vivo transfection ofbronchial,epithelium of rat lung and pig lung with an efficiencycomparable with that of an adenoviral vector has been demonstrated.

[0015] Vectors that utilise integrin receptors to mediate gene transferhave the advantage that they target a large number of different types ofcells in the body as integrin receptors are relatively widespread. Insome circumstances, for example, in in vivo treatment, however, it maybe preferable to target recipient cells more specifically.

[0016] It is an object of the present invention to provide improvedvector complexes with enhanced cell targeting properties. The presentinvention is based on the development of synthetic targeting non-viralvector complexes that carry a ligand that is more cell-type selectivethan the ligands of the prior art.

[0017] Previous approaches to targeted non-viral vectors have includedthe use of antibodies to substances involved in cell-cell adhesion. Forexample, vectors including monoclonal antibodies that target receptorson neuroblastoma cells (Yano et al, 2000) are known. Further examples oftargeting systems have proposed galactose for targeting liver cells (Hanet al. 1999 Bettinger et al. 1999) and asialogylcoprotein, also forliver cells (Wu et al. 1991). However, such methods have been effectiveonly in limited circumstances. For example, antibodies have broadapplicability but they are time-consuming to produce and, by virtue oftheir size, are not as suitable for in vivo administration to anorganism as a small molecule ligand. Furthermore, the methods previouslydescribed do not allow targeting to a cell type for which a ligand isnot yet available.

[0018] In the development of effective targeting vectors it is usefulfor several different target-binding ligands to be available. Effectivetargeted transfection requires not only good targeting but alsoeffective transfer of the vector DNA to the nucleus of the target cell.Even if a ligand is effective in targeting and binding to a target cell,effective gene transfection does not always occur. The reasons for thatare, at present, not clear. Accordingly, there remains a degree ofunpredictability regarding whether a ligand that binds effectively to atarget cell will also bring about effective transfection. It istherefore desirable to have available a “pool” of ligands for anyparticular cell surface receptor from which an effective transfectionligand may be selected. Such selection may take place by means of a genetransfer assay using, for example, a reporter gene, or by any othersuitable means.

[0019] The invention is based on the identification of specific peptidesequences that bind to human airway epithelial (HAE) cells. Theidentified families of HAE cell surface receptor binding componentpeptide motifs mediate specific binding to HAE cells.

[0020] The present invention provides peptide having consisting of orcomprising an amino acid sequence selected from

[0021] a) X¹SM [SEQ.ID.NO.:1];

[0022] b) LX²HK [SEQ.ID.NO.:2];

[0023] c) PSGX³ARA [SEQ.ID.NO.:9];

[0024] d) SX⁴RSMNF (SEQ.ID.NO.:16]; and

[0025] e) LX⁵HKSMP (SEQ.ID.NO.:18],

[0026] in which X¹ is a basic amino acid residue, X² is Q or P, X³ is Aor T, X⁴ is an acidic amino acid residue and X⁵ is P or Q.

[0027] Preferably, the peptide of the invention consists of or comprisesan amino acid sequence selected from

[0028] a) X¹SM (SEQ.ID.NO.:1];

[0029] b) LX²HK [SEQ.ID.NO.:2]; and

[0030] c) PSGAARA (SEQ.ID.NO.:3],

[0031] in which X¹ is a basic amino acid residue and X² is Q or P.

[0032] Preferably X¹ is K or R. Preferably X² is P. Preferably X³ is A.Preferably X⁴ is E or Q [SEQ.ID.No.:17]. More preferably X⁴ is E.Preferably X⁵ is P.

[0033] Preferably, a peptide of the invention comprises a sequenceselected from LQHKSMP [SEQ.ID.NO.:4], LPHKSMP (SEQ.ID.NO.:5), VKSMVTH[SEQ.ID.NO.:6], SERSMNF [SEQ.ID.NO.:7], VGLPHKF [SEQ.ID.NO.:8), YGLPHKF[SEQ.ID.NO.19], PSGAARA [SEQ.ID.NO.:3), SQRSMNF [SEQ.ID.NO.:36] andPSGTARA [SEQ.ID.NO.:38]. Most preferably, the peptide comprises asequence selected from LQHKSMP [SEQ.ID.NO.:4], and LPHKSMP[SEQ.ID.NO.:5].

[0034] A peptide of the invention may be up to 20 amino acids in length,or may be longer. A peptide of the invention generally has at least 5amino acids but may have perhaps fewer. Generally, a peptide of theinvention has any number of amino acids from 6 to 20 inclusive. Thepeptide may have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 amino acids. Generally, it is preferred for a peptide of theinvention to have 15 amino acids or fewer. For example, a peptide of theinvention may have 12 amino acids or fewer. Preferably a peptide of theinvention according to the invention has 10 amino acids or fewer.Generally, it is preferred for a peptide of the invention to have 5 ormore amino acids. For example, a peptide of the invention may have 6 ormore amino acids. For example a peptide of the invention has 7 aminoacids. In the case of a peptide comprising amino acid sequence c) above,the minimum size is 7 amino acids.

[0035] Preferably, a peptide of the invention is such that X¹ is K or Ror X² is Q or P.

[0036] A peptide of the invention may comprise a cyclic region.Preferably, the motif of the invention is flanked by two or morecysteine residues that are capable of forming one or more disulphidebond(s). For example, a peptide of the invention may be “peptide P′”(CLPHKSMPC (SEQ.ID.NO.:10]) or “peptide Q′” (CLQHKSMPC [SEQ.ID.NO.:11]).

[0037] The peptides of the invention find use in HAE cell targetednon-viral transfection vector complexes. They are also useful intargeted viral tranfection vectors.

[0038] The peptide is preferably linked to a polycationic nucleic acidbinding component. The polycationic nucleic acid binding component maybe any polycationic molecule suitable for binding a nucleic acid.

[0039] For example, it may be polyethylenimine. Polyethylenimine (PEI)is a non-toxic, cross linked cationic polymer with gene deliverypotential (Proc. Natl. Acad. Sci., 1995, 92, 7297-7301). For example,the peptide may be linked to the PEI structure via a disulphide bridgeusing methods known in the art (for example, Gene Therapy, 1999, 6,138-145). Polyethylenimine is obtainable from Fluka (800 kDa) or fromSigma (50 kDa) or alternatively pre-diluted for transfection purposesfrom PolyPlus-tranfection (Illkirch, France). Typically, PEI is mostefficient when in a 9 fold excess over DNA (the excess ratio beingcalculated as PEI nitrogen: DNA phosphate) and at pH 5-8. Suchparameters may optimised in a manner familiar to the person skilled inthe art.

[0040] Another example of a nucleic acid-binding polycationic moleculeis an oligopeptide comprising one or more cationic amino acids. Such aoligopeptide may, for example, be an oligo-lysine molecule having from 5to 25 lysine moieties, preferably having from 10 to 20 lysine moieties,for example 16 lysine moieties, an oligo-histidine molecule, or anoligo-arginine molecule or a combined oligomer comprising anycombination of histidine, arginine and lysine residues and having atotal of from 5 to 25 residues, preferably having from 10 to 20residues, for example 16 residues.

[0041] The peptide may be attached to the polycationic nucleic acidbinding component via a spacer.

[0042] A spacer element is generally a peptide, that is to say, itcomprises amino acid residues. The amino acids may be naturallyoccurring or non-naturally occurring. They may have L- orD-configuration. A spacer may have two or more amino acids. It may, forexample, comprise three or more amino acids, for example, four or more,for example, five or more, for example, up to ten amino acids or more.The amino acids may be the same or different, but the use of multiplelysine residues (or other cationic amino acids suitable for use in thepolycationic nucleic acid-binding component of a vector complex) shouldbe avoided in the spacer as oligolysine sequences have activity as apolycationic nucleic acid-binding component of a vector complex of thepresent invention.

[0043] The spacer may be, for example, the dipeptide glycine-glycine(GG) or glycine-alanine (GA). Generally it is preferable that the spaceris longer and/or more hydrophobic than the dipeptide spacers GG and GA.

[0044] The spacer may be more hydrophobic than the dipeptides GG and GA.For example, amino acids that are more hydrophobic than glycine andalanine may be used. Examples of hydrophobic amino acids are well knownand include ε-amino hexanoic acid.

[0045] A spacer may be either longer or more hydrophobic than thedipeptides GG and GA, or it may be both longer and more hydrophobic. Anexample of the latter type of spacer is XSXGA, wherein S=serine,G=glycine, A=alanine and X=ε-amino hexanoic acid. This spacer is highlyhydrophobic.

[0046] The invention further provides a peptide derivative of formulaA-B-C wherein

[0047] A is a polycationic nucleic acid-binding component,

[0048] B is a spacer element, and

[0049] C is a peptide as described above.

[0050] Polycationic nucleic acid-binding component A may be anypolycationic nucleic acid-binding component as described above. Spacerelement B may be any of the spacer elements described above.

[0051] The invention further provides a non-viral transfection complexcomprising:

[0052] (i) a nucleic acid,

[0053] (ii) a lipid component,

[0054] (iii) a polycationic nucleic acid-binding component, and

[0055] (iv) a cell surface receptor binding component, comprising apeptide as described above.

[0056] The cell surface receptor binding component may have the featuresdescribed above in relation to the peptides of the invention.

[0057] The cell surface receptor binding component peptides wereidentified by selection from a peptide library of random 7-mers(peptides having seven amino acid residues) and random 12-mers (peptideshaving twelve amino acid residues) displayed on filamentous phageparticles. Results obtained using the random 7-mer library were betterthan those using the random 12-mer peptide library. The reasons for thedifference in performance of the seven and twelve amino acid library arenot known at present. It is possible that the larger amino acid insertin the phage coat protein reduces the viability of the phage and/or thatthe additional protein synthesis requirement places too great a burdenon the E.coli bacteria. Alternatively, or in addition, impurities in ordefects of the 12-mer library may have adversely affected the outcome ofthe experiments with that library. It appears, however, that smallerpeptides, for example heptameric peptides are preferred. Accordingly,the peptide of the invention preferably has 4 to 11 amino acids, morepreferably 4 to 10 amino acids, for example 7 amino acids.

[0058] The 7-mer library used was a C7C library (i.e. random 7-merpeptides flanked by cysteine residues) obtained from New England BiolabsInc. The 12-mer library used was also obtained from New England BiolabsInc.

[0059] As indicated above, the HAE cell surface receptor bindingpeptides of the invention were identified by selection from a phagedisplay library comprising random peptide sequences seven residues inlength flanked by cysteine residues to allow cyclisation. Such selectionprocedures are generally known. According to such procedures,suspensions of phage are incubated with target cells. Unbound phage arethen washed away and, subsequently, bound phage are extracted either bywashing the remaining cells with a low pH buffer or by lysing the cells.E. coli are then infected with released phage and a preparation of firstround phage is obtained. The cycle is performed repeatedly, for examplethree times and, in order to enrich for targeting phage, the stringencyconditions may be increased in the later rounds of selection, forexample by increasing the number of wash steps, introducing a low pHwash prior to elution and preselecting with wells coated with mediumblocker.

[0060] Following selection by successive rounds of phage amplification,it has been found that phage with high affinity for HAE cells may beselected further by whole cell ELISA using plated HAE cells. Followingincubation of the phage with the HAE cells, the cells are washed andretained phage may then be detected by immunostaining. Cell specificityis assessed by comparing phage binding to target cells with phagebinding to the wells on which the cells were plated and with phagebinding to NIH 3T3 fibroblast control cells.

[0061] Using the whole cell ELISA (Enzyme-Linked ImmunoSorbent Assay)assay described above, high affinity and high specificity bindingpeptides were identified. The cells to which high affinity phage werebound were lysed to release the bound phage particles. The phage DNA wasisolated and sequenced.

[0062] The amino acid sequences of clones obtained from cell lysiseluted C7C phage in a first experiment are shown in Table 1a. TABLE 1aSequence Clone frequency SEQ. ID LQHKSMP 3 4 LPHKSMP 1 5 YGLPHKF 1 19SERSMNF 3 7 VKSMVTH 2 6 PSGAARA 2 3

[0063] The amino acid sequences of clones obtained from cell lysiseluted C7C phage in a second experiment are shown in Table 1b. TABLE 1bSequence Clone Frequency SEQ. ID. NO. SERSMNF 18 7 YGLPHKF 12 19 PSGAARA9 3 LQHKSMP 3 4 VKSMVTH 3 6 SQRSMNF 2 36 QPLRHHQ 2 37 LPHKSMP 1 5PSGTARA 1 38 KQRPAWL 1 39 IPMNAPW 1 40 SLPFARN 1 41 GPARISF 1 42 MGLPLRF1 43

[0064] The 56 sequenced clones from the third round of panning ofHAEo-in the second experiment were represented by the 14 sequences shownin Table 1b, with some sequences being represented by multiple phageclones. The sequences shown were each flanked by two cyteine residues inthe phage and are thus constrained in a loop formation by disulphidebonds between them. For the avoidance of doubt, all of the sequences inTables 1a and 1b form part of the present invention.

[0065] An analysis of the motifs found in the positive clone amino acidsequences of Table 1a (the first experiment) is shown in

[0066] Table 2a. TABLE 2a Motif Motif Sequence SEQ. ID. Clone frequencyfrequency KSM/RSM LQHKSMP 4 3 9 LPHKSMP 5 1 VKSMVTH 6 2 SERSMNF 7 3 LXHKLQHKSMP 4 3 5 LPHKSMP 5 1 YGLPHKF 19 1 LXHKSMP LQHKSMP 4 3 4 LPHKSMP 5 1PSGAARA* PSGAARA 3 2 2

[0067] An analysis of the motifs found in the positive clone amino acidsequences of Table 1b (the second experiment) is shown in Table 2b.TABLE 2b Motif Motif Sequence SEQ. ID. Clone frequency frequency KSM/RSMSERSMNF 7 18 27 SQRSMNF 36 2 VKSMVTH 6 3 LQHKSMP 4 3 LPHKSMP 5 1 SXRSMNFSERSMNF 7 18 20 SQRSMNF 36 2 LXHK LQHKSMP 4 3 16 LPHKSMP 5 1 YGLPHKF 1912 PSGXARA PSGAARA 3 9 10 PSGTARA 38 1 LXHKSMP LPHKSMP 5 3 4 LQHKSMP 4 1

[0068] The sequences found in the first experiment (Table 1a) werecompared and ranked for their binding strength by ELISA using a range ofphage titres (Table 3). In Table 3, the sequences are ranked in order ofbinding affinity to HAE cells. It was found that the sequence LPHKSMP(“Peptide P”) had the highest binding affinity. TABLE 3 Clone SequenceSEQ. ID frequency Motifs LPHKSMP 5 1 LXHK, LXHKSMP, KSM LQHKSMP 4 3LXHK, LXHKSMP, KSM YGLPHKF 19 1 LXHK VKSMVTH 6 2 KSM PSGAARA 3 2 PSGAARASERSMNF 7 3 RSM

[0069] From the Tables it may be seen that the motifs KSM/RSM and LXHKwere present in several of the clones. This strongly suggests that thosemotifs are important for HAE cell surface binding. It is at present notknown to which HAE receptor(s) the sequences bind. The various motifsmay target the same receptor or they may target different receptors.

[0070] Good binding indicates a high affinity interaction and/or thebinding of a cell surface receptor molecule present in high numbers onthe cell surface. The LPHK version of the LXHK motif provides betterbinding than the LQHK version and the KSM version of the XSM motifprovides better binding than the RSM version. The LXHK motif and the KSMmotif are frequently found together. This may be due to a cooperativeeffect, possibly due to the motifs binding to two cell surface receptormolecules.

[0071] Although the peptide sequences of the invention were identifiedusing HAE cells, their utility is not limited to use with HAE cells. Thereceptors to which the peptides bind may be expressed in other celltypes. Cell types with which peptides of the invention may be used maybe identified by any suitable screening procedure.

[0072] The transfection properties the vector complexes of the inventionwere investigated in HAE cell transfection experiments as describedbelow.

[0073] Non-viral transfection vector complexes incorporating theidentified sequences were prepared. Peptides were synthesised usingstandard solid phase synthetic chemistry and a sixteen-lysine tail wasadded. The most frequently occurring peptides were chosen for synthesis,with peptide LPHKSMP chosen because it contains three motifs. Eachpeptide was assigned a single letter name. The peptides chosen forsynthesis are shown in Table 4. TABLE 4 SEQ. Clone Peptide Sequence ID.frequency Motifs E SERSMNF 7 18 RSM, SXRSMNF Y YGLPHKF 19 12 LXHK GPSGAARA 3 9 PSGXARA V VKSMVTH 6 3 KSM Q LQHKSMP 4 3 LXHK, LXHKSMP, KSM PLPHKSMP 5 1 LXHK, LXHKSMP, KSM

[0074] Luciferase reporter gene DNA was used as the transfection DNA.Transfection complexes were made by mixing the components in theorder 1) lipid, then 2) peptide and, finally 3) DNA, followed bydilution. The vector complex suspension was applied to HAE cells andcontrol cells. Vector complexes incorporating peptide Q([K]₁₆-GACLQHKSMPCG [SEQ.ID.NO.:12]) and vector complexes incorporatingpeptide P ([K]₁₆-GACLPHKSMPCG [SEQ.ID.NO.:13]) were synthesised andcompared with vector complexes incorporating peptide S([K]₁₆-GACYKHPGFLCG] [SEQ.ID.NO.:14]) which is a control peptide havingthe same amino acid constituents as peptide P but in a randomised order(the “scambled control”), Peptide 12 ([K]₁₆-XSXGACRRETAWACG[SEQ.ID.NO.:15]), a targeting peptide known to bind to alpha 5 beta 1integrins and Peptide K ([K]₁₆) a DNA binding moiety with no targetingligand attached.

[0075] Transfections of HAE cells and 3T3 cells were performed in 96well plates containing 20,000 cells plated 24 hours earlier. In thetransfection vector complex, peptide to DNA charge ratios (±) were usedat 1.5:1, 3:1 and 7:1. At physiological pH, DNA carries negative chargeand the polycationic-nucleic acid binding component carries positivecharge. The “charge ratio” is accordingly the ratio of the charges ofthe two components in the complex. The lipid component was maintained ata constant proportion, by weight, relative to DNA of 0.75:1. The resultsof the transfection experiments are shown in FIG. 3.

[0076] At a 7:1 charge ratio, the transfection efficiency of vectorcomplexes containing peptide P was five-fold higher than the next bestpeptide, peptide 12 at a 3:1 charge ratio. Peptide P was 150-fold betterthan peptide S (the scrambled control) at the charge ratio of 7:1indicating that the transfection efficiency was receptor specific.Vector complexes containing peptide P were almost nine-fold moreefficient those containing peptide K, again indicating receptorspecificity. The fact that vector complexes containing peptide Kperformed better in the assay than vector complexes containing peptide Ssuggests that steric hindrance by the scrambled motif in peptide S mayplay a role.

[0077] Despite the similar HAE cell surface binding properties ofpeptide P and peptide Q (See FIG. 2), peptide P performed significantlybetter than peptide Q in the transfection assay. This result suggeststhat binding properties alone are not sufficient to achieve highefficiency of transfection.

[0078] The HAE cell surface receptor binding peptide component for usein the vector complex of the invention may be synthesised using standardsolid phase peptide synthesis methods.

[0079] The identity of the molecules bound by the peptides used intransfections was explored by carrying out a BLAST search (Tables 5a and5b). Homologies were found to several molecules of interest which maybind molecules present on the surface of epithelial cells in the lung.Pathogen peptides with homology with the peptides of the invention areshown in Table 5a, whilst cell adhesion molecules with homology with thepeptides of the invention are shown in Table 5b. TABLE 5a PeptideHomology Protein Pathogen Receptor LPHKSMP/ LHKSM Glyco- Human Cellsurface LQHKSMP protein B herpesvirus heparan sulphate SXRSMNF SDRSMNCapsid Human ICAM-1 or LDL binding rhinovirus receptor family proteinVP2 YGLPHKF YGLPHK Unknown Legionella Unknown pneumophila epithelialcell receptors VKSMVTH VKSMITQ Adhesin Mycoplasma Cell surface P1Pneumoniae sialoglycoproteins

[0080] TABLE 5b Peptide Homology Protein Species Receptor SXRSMNF SERSMNSelectin Rat Cell surface glycoproteins ERSMDF Laminin, alpha HumanExtracellular 5 matrix components including integrins LXHKSMP LPHKNMEpithelial Mouse/ Dimerises, also caderin rabbit binds integrin a-E,(ovumorulin) b-7

[0081] Epithelial cadherin is a molecule which is involved in cell-celladhesion and forms complexes with β-catenin. Human herpesvirusglycoprotein B binds cell surface heparan sulphate proteoglycans.Selectin binds cell surface glycoproteins. Laminin, alpha 5 is abasement membrane protein found in epithelium. The capsid bindingprotein VP2 of the rhinovirus binds ICAM-1 or the LDL receptor family ofmolecules in the upper respiratory tract. P-glycoprotein is a molecularpump molecule which is localised in the cell membrane, and coagulationfactor XII has been shown to bind cytokeratins on epithelial cells.

[0082] In so far as any motif or any peptide of the invention occurs ina naturally-occurring protein, the peptides of invention do not includesuch a naturally-occurring full-length protein. Generally, the peptidesof the invention are 100 or fewer amino acids in length; preferably thepeptides of the invention are 50 or fewer amino acids in length.Typically, they are of sizes described above.

[0083] The peptides of the invention finds utility in the study ofconditions involving the pathogens and the cell adhesion molecules givenin Tables 4a and 4b. They are also useful in the development oftreatments for those conditions.

[0084] The nucleic acid component may be obtained from natural sources,or may be produced recombinantly or by chemical synthesis. It may bemodified, for example, to comprise a molecule having a specificfunction, for example, a nuclear targeting molecule. The nucleic acidmay be DNA or RNA. DNA may be single stranded or double stranded. Thenucleic acid may be suitable for use in gene therapy, in genevaccination or in anti-sense therapy. The nucleic acid may be or mayrelate to a gene that is the target for particular gene therapy or maybe a molecule that can function as a gene vaccine or as an anti-sensetherapeutic agent. The nucleic acid may be or correspond to a completecoding sequence or may be part of a coding sequence.

[0085] Alternatively, the nucleic acid may encode a protein that iscommercially useful, for example industrially or scientifically useful,for example an enzyme; that is pharmaceutically useful, for example, aprotein that can be used therapeutically or prophylactically as amedicament or vaccine; or that is diagnostically useful, for example, anantigen for use in an ELISA. Host cells capable of producingcommercially useful proteins are sometimes called “cell factories”.

[0086] Appropriate transcriptional and translational control elementsare generally provided. For gene therapy, the nucleic acid component isgenerally presented in the form of a nucleic acid insert in a plasmid orvector. In some cases, however, it is not necessary to incorporate thenucleic acid component in a vector in order to achieve expression. Forexample, gene vaccination and anti-sense therapy can be achieved using anaked nucleic acid.

[0087] The nucleic acid is generally DNA but RNA may be used in somecases, for example, in cancer vaccination. The nucleic acid componentmay be referred to below as the plasmid component or component “D”.

[0088] As indicated above, the polycationic nucleic acid-bindingcomponent is any polycation that is capable of binding to DNA or RNA.The polycation may have any number of cationic monomers provided theability to bind to DNA or RNA is retained. For example, from 3 to 100cationic monomers may be present, for example, from 10 to 20, forexample from 14 to 18, especially about 16. An oligolysine isparticularly preferred, for example, having from 10 to 20 lysineresidues, for example, from 13 to 19, for example, from 14 to 18, forexample, from 15 to 17 residues, especially 16 residues i.e. [K]₁₆, “K”denoting lysine.

[0089] A further preferred cationic polymer is polyethylenimine (Proc.Natl. Acad. Sci., 1995, 92, 7297-7301).

[0090] The polycationic DNA-binding or RNA-binding component mayadvantageously be linked or otherwise attached to the cell surfacereceptor-binding component. A combined cell surface receptor-bindingcomponent/polycationic DNA-binding or RNA-binding component may bereferred to below as component “I”. For example, a polycationicDNA-binding or RNA-binding component may be chemically bonded to a cellsurface receptor-binding component, for example, by a peptide bond inthe case of an oligolysine. The polycationic component may be linked atany position of the cell surface receptor-binding component. Preferredcombinations of cell surface receptor-binding component and polycationicDNA-binding or RNA-binding component are an oligolysine, especially[K]₁₆, linked via a peptide bond to a peptide, for example, a peptide asdescribed above. A further preferred combination of cell surfacereceptor-binding component and polycationic DNA-binding or RNA-bindingcomponent are a polyethylenimine linked via a covalent link to apeptide, for example, a peptide as described above. For example such acovalent link may be a disulphide bridge or a succinimidyl bridge.

[0091] The lipid component may be or may form a cationic liposome. Thelipid component may be or may comprise one or more lipids selected fromcationic lipids and lipids having membrane destabilising or fusogenicproperties, especially a combination of a cationic lipid and a lipidthat has membrane destabilising properties.

[0092] A preferred lipid component (“L”) is or comprises the neutrallipid dioleyl phosphatidylethanolamine, referred to herein as “DOPE”.DOPE has membrane destabilising properties sometimes referred to as“fusogenic” properties (Farhood et al. 1995). Other lipids, for example,neutral lipids, having membrane destabilising properties, especiallymembrane destabilising properties like those of DOPE may be used insteadof or as well as DOPE.

[0093] Other phospholipids having at least one long chain alkyl group,for example, di(long alkyl chain)phospholipids may be used. Thephospholipid may comprise a phosphatidyl group, for example, aphosphatidylalkanolamine group, for example, a phosphatidyl-ethanolaminegroup.

[0094] A further preferred lipid component is or comprises the cationiclipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride,referred to herein as “DOTMA”. DOTMA has cationic properties. Othercationic lipids may be used in addition to or as an alternative toDOTMA, in particular cationic lipids having similar properties to thoseof DOTMA. Such lipids are, for example, quaternary ammonium saltssubstituted by three short chain alkyl groups, and one long chain alkylgroup. The short chain alkyl groups may be the same or different, andmay be selected from methyl and ethyl groups. At least one and up tothree of the short chain alkyl group may be a methyl group. The longalkyl chain group may have a straight or branched chain, for example, adi(long chain alkyl)alkyl group.

[0095] Another preferred lipid component is or comprises the lipid2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoridoacetate,referred to herein as “DOSPA”. Analogous lipids may be used in additionto or as an alternative to DOSPA, in particular lipids having similarproperties to those of DOSPA. Such lipids have, for example, differentshort chain alkyl groups from those in DOSPA.

[0096] A preferred lipid component comprises DOPE and one or more otherlipid components, for example, as described above. Especially preferredis a lipid component that comprises a mixture of DOPE and DOTMA. Suchmixtures form cationic liposomes. An equimolar mixture of DOPE and DOTMAis found to be particularly effective. Such a mixture is knowngenerically as “lipofectin” and is available commercially under the name“Lipofectin”. The term “lipofectin” is used herein generically to denotean equimolar mixture of DOPE and DOTMA. Other mixtures of lipids thatare cationic liposomes having similar properties to lipofectin may beused. Lipofectin is particularly useful as it is effective in all celltypes tested.

[0097] A further preferred lipid component comprises a mixture of DOPEand DOSPA. Such mixtures also form cationic liposomes. A mixture of DOPEand DOSPA in a ratio by weight 3:1 DOSPA:DOPE is particularly effective.Such a mixture, in membrane filtered water, is available commerciallyunder the name “Lipofectamine”. Mixtures comprising DOPE, DOTMA andDOSPA may be used, for example, mixtures of lipofectin andlipofectamine.

[0098] Other cationic lipids are available commercially, for example,DOTAP (Boehringer-Mannheim) and lipids in the Tfx range (Promega). DOTAPis N-[1-(2,3-diolyloxy)propyl]-N,N,N-trimethylammonium methylsulphate.The Tfx reagents are mixtures of a synthetic cationic lipid[N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammoniumiodide and DOPE. All the reagents contain the same amount of thecationic lipid component but contain different molar amounts of thefusogneic lipid, DOPE.

[0099] However, lipofectin and lipofectamine appear to be markedly moreeffective as the lipid component in LID vector complexes of the presentinvention than are DOTPA and Tfx agents.

[0100] The effectiveness of a putative cell surface receptor-bindingcomponent, polycationic DNA-binding or RNA-binding component, or oflipid component or of any combination thereof may be determined readilyusing the methods described herein.

[0101] The efficiency of transfection using a transfection complex asdescribed above as transfection vector is influenced by the ratio lipidcomponent:cell surface receptor-binding component:DNA or RNA. For anychosen combination of components for any particular type of cell to betransfected, the optimal ratios can be determined simply by admixing thecomponents in different ratios and measuring the transfection rate forthat cell type, for example, as described herein.

[0102] Lipofectin and lipofectamine appear to be particularly effectivein enhancing transfection in the system described above. Lipofectin hasthe advantage that only very small amounts are required. Any sideeffects that may occur are therefore minimised. A suitable weight ratiobetween the lipid and the DNA components has been found to be 0.75:1.For any given transfection experiment, this ratio may be optimised usingmethods known in the art.

[0103] Cells that may be transfected by a transfection vector complexincorporating a peptide of the invention include, for example,endothelial or epithelial cells, for example, cells of the any part ofthe airway epithelium, including bronchial and lung epithelium, and thecorneal endothelium. The airway epithelium is an important target forgene therapy for cystic fibrosis and asthma.

[0104] A transfection vector complex as described above may be producedby admixing components (i), (ii), (iii) and (iv).

[0105] Although the components may be admixed in any order, it isgenerally preferable that the lipid component is not added last. In thecase where there is a combined cell surface receptor-bindingcomponent/polycationic DNA-binding or RNA-binding component it isgenerally preferable to combine the components in the following order:lipid component; combined cell surface receptor-binding/polycationicDNA-binding or RNA-binding component; DNA or RNA component, for example,in the order: lipofectin, oligolysine-peptide component, DNA or RNAcomponent.

[0106] A transfection mixture comprising a cell surface receptor-bindingcomponent, a polycationic nucleic acid-binding component, and a lipidcomponent may be used to produce a nucleic acid-containing transfectionvector complex as described above by the incorporation of a nucleic acidwith the mixture, for example, by admixture. Alternatively, thetransfection mixture may be used for the production of a vector complexwhich comprises, instead of the nucleic acid component, any othercomponent that is capable of binding to the polycationic nucleic-acidbinding component, for example, a protein.

[0107] The individual components of a transfection mixture of theinvention are each as described above in relation to the transfectionvector complex. The preferred components, preferred combinations ofcomponents, preferred ratios of components and preferred order ofmixing, both with regard to the mixture and to the production of avector complex, are as described above in relation to the transfectionvector complex.

[0108] A transfection mixture preferably comprises an equimolar mixtureof DOPE and DOTMA (lipofectin) as the lipid component and anoligolysine-peptide especially a [K]₁₆-peptide as a combined cellsurface receptor-binding component/nucleic acid-binding component. Thepreferred molar ratio lipofectine:oligolysine-peptide is 0.75:4.

[0109] The invention further provides a non-viral transfection complexcomprising:

[0110] (i) a nucleic acid,

[0111] (iii) a polycationic nucleic acid-binding component, and

[0112] (iv) a cell surface receptor binding component, comprising apeptide as described above.

[0113] The cell surface receptor binding component may have the featuresdescribed above in relation to the peptides of the invention. Thenucleic acid component and the polycationic nucleic acid-bindingcomponent may be as described above in relation to the non-viraltransfection complex comprising (i), (ii), (iii) and (iv).

[0114] The effectiveness of a putative cell surface receptor-bindingcomponent and polycationic DNA-binding or RNA-binding component may bedetermined readily using the methods described herein.

[0115] The efficiency of transfection using a transfection complex asdescribed above as transfection vector is influenced by the ratio ofcell surface receptor-binding component: polycationic nucleicacid-binding component: DNA or RNA. For any chosen combination ofcomponents for any particular type of cell to be transfected, theoptimal ratios can be determined simply by admixing the components indifferent ratios and measuring the transfection rate for that cell type,for example, as described herein.

[0116] Cells that may be transfected by a transfection vector complexincorporating a peptide of the invention include, for example,endothelial or epithelial cells, for example, cells of any part of theairway epithelium, including bronchial and lung epithelium, and thecorneal endothelium. The airway epithelium is an important target forgene therapy for cystic fibrosis and asthma.

[0117] A transfection vector complex as described above may be producedby admixing components (i),(iii) and (iv).

[0118] Although the components may be admixed in any order, it isgenerally preferable to combine the components in the following order:combined cell surface receptor-binding/polycationic DNA-binding orRNA-binding component; DNA or RNA component, for example, in the order:polyethylenimine-peptide component; DNA or RNA component.

[0119] A transfection mixture comprising a cell surface receptor-bindingcomponent and a polycationic nucleic acid-binding component may be usedto produce a nucleic acid-containing transfection vector complex asdescribed above by the incorporation of a nucleic acid with the mixture,for example, by admixture. Alternatively, the transfection mixture maybe used for the production of a vector complex which comprises, insteadof the nucleic acid component, any other component that is capable ofbinding to the polycationic nucleic-acid binding component, for example,a protein.

[0120] The individual components of a transfection mixture of theinvention are each as described above in relation to the transfectionvector complex. The preferred components, preferred combinations ofcomponents, preferred ratios of components and preferred order ofmixing, both with regard to the mixture and to the production of avector complex, are as described above in relation to the transfectionvector complex.

[0121] The present invention also provides a process for expressing anucleic acid in host cells, which comprises contacting the host cells invitro or in vivo with a receptor-targeted vector complex of theinvention comprising the nucleic acid and then culturing the host cellsunder conditions that enable the cells to express the nucleic acid.

[0122] The present invention further provides a process for theproduction of a protein in host cells, which comprises contacting thehost cells in vitro or in vivo with a receptor-targeted vector complexof the invention that comprises a nucleic acid that encodes the protein,allowing the cells to express the protein, and obtaining the protein.The protein may be obtained either from the host cell or from theculture medium.

[0123] The present invention further provides a method of transfectingcells comprising subjecting the cells to a vector complex according tothe invention.

[0124] The invention further provides cells, transfected with a nucleicacid by a method according to the invention, and also the progeny ofsuch cells.

[0125] The present invention further provides a disease model for use intesting candidate pharmaceutical agent, which comprises cellstransfected by a method according to the invention with a nucleic acidsuitable for creating the disease model.

[0126] The present invention also provides a pharmaceutical compositionwhich comprises a receptor-targeted vector complex of the inventioncomprising a nucleic acid in admixture or conjunction with apharmaceutically suitable carrier. The composition may be a vaccine.

[0127] The present invention also provides a method for the treatment orprophylaxis of a condition caused in a human or in a non-human animal bya defect and/or a deficiency in a gene, which comprises administering tothe human or to the non-human animal a receptor-targeted vector complexof the invention comprising a nucleic acid suitable for correcting thedefect or deficiency.

[0128] The present invention also provides a method for therapeutic orprophylactic immunisation of a human or of a non-human animal, whichcomprises administering to the human or to the non-human animal areceptor-targeted vector complex of the invention comprising anappropriate nucleic acid.

[0129] The present invention also provides a method of anti-sensetherapy of a human or of a non-human animal, comprising anti-sense DNAadministering to the human or to the non-human animal areceptor-targeted vector complex of the invention comprising theanti-sense nucleic acid.

[0130] The present invention also provides the use of areceptor-targeted vector complex of the invention comprising a nucleicacid for the manufacture of a medicament for the prophylaxis of acondition caused in a human or in a non-human animal by a defect and/ora deficiency in a gene, for therapeutic or prophylactic immunisation ofa human or of a non-human animal, or for anti-sense therapy of a humanor of a non-human animal.

[0131] A non-human animal is, for example, a mammal, bird or fish, andis particularly a commercially reared animal.

[0132] The nucleic acid, either DNA or RNA, in the vector complex isappropriate for the intended use, for example, for gene therapy, genevaccination, or anti-sense therapy. The DNA or RNA and hence the vectorcomplex is administered in an amount effective for the intended purpose.

[0133] The treatments and uses described above may be carried out byadministering the respective vector complex, agent or medicament in anappropriate manner, for example, administration may be topical, forexample, in the case of airway epithelia.

[0134] In a further embodiment, the present invention provides a kitcomprising a receptor-targeted vector complex of the inventioncomprising a nucleic acid.

[0135] The present invention also provides a kit that comprises thefollowing items: (a) a cell surface receptor-binding component; (b) apolycationic nucleic acid-binding component, and (c) a lipid component.Such a kit may further comprise (d) a nucleic acid. Such a nucleic acidmay be single-stranded or double stranded and may be a plasmid or anartificial chromosome. The nucleic acid component may be provided by avector complex suitable for the expression of the nucleic acid, thevector complex being either empty or comprising the nucleic acid. For invitro purposes, the nucleic acid may be a reporter gene. For in vivotreatment purposes, the nucleic acid may comprise DNA appropriate forthe correction or supplementation being carried out. Such DNA may be agene, including any suitable control elements, or it may be a nucleicacid with homologous recombination sequences. It has been found thatpeptide/DNA/lipid/polycationic nucleic acid-binding component complexesare especially stable in salt free buffer (for example in water, or 5%dextrose).

[0136] The present invention also provides a kit that comprises thefollowing items: (a) a cell surface receptor-binding component; and (b)a polycationic nucleic acid-binding component. Such a kit may furthercomprise (d) a nucleic acid. Such a nucleic acid may be single-strandedor double stranded and may be a plasmid or an artificial chromosome. Thenucleic acid component may be provided by a vector complex suitable forthe expression of the nucleic acid, the vector complex being eitherempty or comprising the nucleic acid. The nucleic acid component may beprovided by a vector complex suitable for the expression of the nucleicacid, the vector complex being either empty or comprising the nucleicacid. For in vitro purposes, the nucleic acid may be a reporter gene.For in vivo treatment purposes, the nucleic acid may comprise DNAappropriate for the correction or supplementation being carried out.Such DNA may be a gene, including any suitable control elements, or itmay be a nucleic acid with homologous recombination sequences. It hasbeen found that peptide/DNA/polycationic nucleic acid-binding componentcomplexes are especially stable in salt free buffer (for example inwater, or 5% dextrose).

[0137] The components (a) to (d) kit are, for example, as describedabove in relation to a cell surface receptor-targeted transfectionvector complex or a mixture as described above.

[0138] A kit generally comprises instructions, which preferably indicatethe preferred ratios of the components and the preferred order of use oradmixing of the components, for example, as described above. A kit maybe used for gene therapy, gene vaccination or anti-sense therapy.Alternatively, it may be used for transfecting a host cell with anucleic acid encoding a commercially useful protein i.e. to produce aso-called “cell factory”.

[0139] In a kit of the invention the components including the preferredcomponents are, for example, as described above in relation to a vectorcomplex of the present invention.

[0140] The polycationic nucleic acid binding component is preferably anoligolysine, as described above. The lipid component is preferablycapable of forming a cationic liposome, and preferably is or comprisesDOPE and/or DOTMA, for example, an equimolar mixture thereof, or is orcomprises DOSPA, for example, a mixture of DOPE and DOSPA, for examplein the weight ratio DOPE:DOSPA of 1:3. The rations between thecomponents are preferably as described above, as is the order of mixingof the components.

[0141] Targets for gene therapy are well known and include monogenicdisorders, for example, cystic fibrosis, various cancers, andinfections, for example, viral infections, for example, with HIV. Forexample, transfection with the p53 gene offers great potential forcancer treatment. Targets for gene vaccination are also well known, andinclude vaccination against pathogens for which vaccines derived fromnatural sources are too dangerous for human use and recombinant vaccinesare not always effective, for example, hepatitis B virus, HIV, HCV andherpes simplex virus. Targets for anti-sense therapy are also known.Further targets for gene therapy and anti-sense therapy are beingproposed as knowledge of the genetic basis of disease increases, as arefurther targets for gene vaccination. The present invention enhances thetransfection efficiency and hence the effectiveness of the treatment.

[0142] Vector complexes of the invention may be effective forintracellular transport of very large DNA molecules, for example, DNAlarger than 125 kb, which is particularly difficult using conventionalvectors. This enables the introduction of artificial chromosomes intocells.

[0143] Transfection of the airways, for example, the bronchialepithelium demonstrates utility for gene therapy of, for example,respiratory diseases, such as cystic fibrosis, emphysema, asthma,pulmonary fibrosis, pulmonary hypertension and lung cancer.

[0144] Cystic fibrosis (CF) is the most common monogenic disorder in theCaucasian population. Morbidity is mainly associated with lung disease.CF is caused by mutations in the gene encoding the cystic fibrosistransmembrane conductance regulator protein (CFTR), a cell membranechannel that mediates secretion of chloride ions. Correction of thisdefect in the bronchial cells by CFTR gene transfer will correct thebiochemical transport defect and, hence, the lung disease. Clinicaltrials so far have generated encouraging data but highlighted the needfor more efficient, non-toxic vectors.

[0145] The enhanced levels of transfection make the method of theinvention particularly suitable for the production of host cells capableof producing a desired protein, so-called “cell factories”. Forlong-term production, it is desirable that the introduced nucleic acidis incorporated in the genome of the host cell, or otherwise stablymaintained. That can be readily ascertained. As indicated above, therange of proteins produced in this way is large, including enzymes forscientific and industrial use, proteins for use in therapy andprophylaxis, immunogens for use in vaccines and antigens for use indiagnosis.

[0146] Accordingly, the present invention provides a method of testingdrugs in a tissue model for a disease, wherein the tissue modelcomprises transgenic cells obtained by transfecting cells with a nucleicacid by contacting the cell with a receptor-targeted vector complex ofthe invention comprising a nucleic acid.

[0147] The present invention is especially useful with a receptortargeted vector complex that is capable of high efficiency transfection.In a preferred embodiment, the vector complex comprises four modularelements; an oligolysine, especially [K]₁₆, DNA-binding or RNA-bindingelement; a high affinity cell surface receptor-binding peptide, forexample, a peptide described herein; a DNA or RNA sequence, optionallyin a plasmid, and optionally regulated by a viral promoter and anenhancing element; the cationic liposome DOTMA/DOPE (lipofectin). Thecombination of oligolysine-peptide/DNA or RNA complex with the cationicliposome formulation DOTMA/DOPE is a potent combination. Alternatively aDOPE/DOSPA formulation may be used instead of or in addition to aDOTMA/DOPE formulation. The optimisation of variables associated withcomplex formation and the mode of transfection by LID vector complexeshas been demonstrated.

[0148] The most important variables in the formation of optimal LIDtransfection complexes appear to be the ratio of the three componentsand their order of mixing.

[0149] The invention further provides a method for identifying a cellsurface receptor binding ligand for use in a non-viral transfectionvector complex comprising the steps:

[0150] a) selecting phage from a phage peptide library according totheir binding affinity to cells of interest by bringing the phage intocontact with the cells of interest and washing away non-binding phageand then extracting bound phage particles,

[0151] b) repeating step (a) if necessary, and preferably

[0152] c) selecting from the phage obtained in steps a) and b) thosephage which bind to the cell of interest with high affinity using awhole cell ELISA.

[0153] Preferably, the stringency of the wash in step a) is increasedafter the first round of selection by washing at low pH by washingmultiple times.

[0154] The following non-limiting Examples illustrate the presentinvention. The Examples refer to the accompanying drawings, in which:

[0155]FIG. 1 shows the enhancement of phage binding to HAE cells insuccessive rounds of selection for the C7C library and the 12mer peptidelibrary starting materials.

[0156]FIG. 2 shows the binding specificity of individual phage clones ina whole cell ELISA assay. Binding affinity to HAE cells, to 3T3 controlcells and to the ELISA plate are shown.

[0157]FIG. 3 shows the relative efficiency of transfection of HAE cellsachieved by transfection complexes according to the invention.

[0158]FIG. 4 shows the relative efficiency of transfection of HAE cellsachieved by transfection complexes according to the invention andscrambled control peptides.

[0159]FIG. 5 shows the relative efficiency of transfection of Neuro-2Acells achieved by transfection complexes according to the invention anda control peptide, peptide 6.

[0160]FIG. 6 shows the relative efficiency of transfection of IMR32cells achieved by transfection complexes according to the invention anda control peptide, peptide 6.

[0161]FIG. 7 shows the relative efficiency of transfection of rabbitadventitial cells achieved by transfection complexes according to theinvention and a control peptide, peptide 6.

[0162]FIG. 8 shows the relative efficiency of transfection of 3T3 cellsachieved by transfection complexes according to the invention and acontrol peptide, peptide 6.

EXAMPLES

[0163] Materials & Methods

Example 1

[0164] Peptide Library

[0165] The peptide library used in this study, C7C, was obtained fromNew England Biolabs Inc. Phage growth, titration and amplificationprocedures were performed as described in the manufacturer's handbook.The library consisted of random peptide sequences seven residues inlength and flanked by cystine residues to allow cyclisation by oxidationin air. The library is likely to contain at least 1×10⁹ different aminoacid sequences.

[0166] Selection of Phage from the Library

[0167] HAE cells were grown to confluence in 24-well plates. The RAEcells used were 1HAEo-cells obtained as a gift from Dr. Dieter Gruenertof the University of California, San Francisco (now of the University ofVermont). Cells were washed twice in Tris-buffered saline, pH 7.4 (TBS)before bloacking cells with 2 ml 2% Marvel, 5% bovine serum albumin(BSA)-TBS per well for 30 minutes at 4° C. The blocker was removed and2×10¹¹ phage were added in 1 ml of 2% Marvel, 5% BAS-TBS. The phage wereallowed to bind for 2 hours with shaking at 4° C. before washing fivetiems with 2% BSA-TBS and 5 minutes shaking at 4° C. followed by anotherfive washes with 2% BSA-TBS for a few seconds only. Phage were eluted bythe addition of 400 μl 76 mM citrate buffer pH 2.5 to the wells for 10minutes with shaking at 4° C. The eluate was removed and neutralisedwith 600 μl 1M Tris buffer pH 7.5 and retained as the eluted fraction.The remaining cells were lysed with 1 ml 30 mM Tris buffer pH 8.0, 1 mmEDTA for 1 hour on ice. The cells were scraped from the plate, theeluate transferred to a microcentrifuge tube, and vortexed briefly. Thateluate was retained as the cell-associated fraction. The above describedprocess was repeated three times. In the second and third rounds, thestringency of selection was increased by introduction of preselectionsteps to remove phage that bind to the plastic or to components in themedium and by increasing the number of washes following phage binding.The number of phage present in each eluate (in plaque forming units,PFU) is shown in FIG. 1.

[0168] Whole Cell HAE Cell Binding ELISA

[0169] Binding of phage to tissue cultured HAE cells was investigated bywhole cell ELISA. Approx. 8×10⁴ HAE cells in 100 ml Hanks Balanced SaltsSolution (HBSS) were added to each well of a 96 well plate and incubatedat 37° C. until cells had adhered. The cells were washed gently in HBSSbefore blocking by the addition of 0.5% BSA in HBSS for 30 mins. 1×10¹⁰phage particles in blocker solution were added to each well and allowedto bind at room temperature for 40 minutes. Unbound phage were removedby washing twice with HBSS, and bound phage were fixed to the cells byincubation in 3.7% paraformaldehyde for 10 mins. Cells were washed inPBS and incubated in blocking buffer for 45 mins, followed by threewashes in PBS. Bound phage were detected by the addition of horseradishperoxidase (HRP)-conjugated anti-M13 antibody diluted 1:5000 in blockingbuffer for 1 hour, before washing three times in PBS and developing theELISA with 2, 2′-azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) (ABTS)substrate solution and reading the absorbance on a plate-readingspectrophotometer at 405 nm. The experiment was repeated using 3T3 cellsand using empty wells and the comparison of binding affinities enabledthe identification of phage that bound selectively to HAE cells. Theresults for selected peptides are shown in FIG. 2.

[0170] Peptide-encoding DNA of 12 phage clones that displayed high RAEcell avidity and specificity were sequenced and the peptide sequencededuced. The sequences deduced are shown in table 1. Three major peptidemotifs, KSM/RSM, LXHK and LXHKSMP were identified amongst the 12sequences and one sequence, PSGAARA that contained none of the otherthree motifs. The sequences were compared and ranked for their bindingstrength by ELISA using a range of phage titres (table 3). It was foundthat the sequence LPHKSMP (peptide P) had the highest binding affinity.This sequence and the closely related peptide LQHKSMP (peptide Q) and acontrol, scrambled version of peptide P were selected for transfectionexperiments.

[0171] Peptide Synthesis

[0172] The following oligolysine-peptides were prepared for transfectionexperiments:

[0173] Peptide P: [K]₁₆-GACLPHKSMPCG—binds to HAE cells

[0174] Peptide Q: [K]₁₆-GACLQHKSMPCG—binds to HAE cells

[0175] Peptide S: [K]₁₆-GACYKHPGFLCG—non-binding control

[0176] Peptide 12: [K]₁₆-XSXGACRRETAWACG—binds to alpha 5 beta 1integrins (X=ε-amino hexanoic acid).

[0177] K₁₆: DNA binding moiety, no targeting ligand.

[0178] The oligolysine-peptides were synthesised using standard solidphase oligopeptide synthesis methods.

[0179] Transfection Experiments

[0180] Peptides identified from phage that displayed desirable cellbinding characteristics were synthesised using standard solid-phasepeptide synthetic chemistry and a sixteen-lysine tail was attached usingstandard synthesis methods. Control peptide (S), consisted of the sameamino acid constituents as the targeting peptide P but in a randomisedorder, was synthesised for incorporation into lipopolyplex formulations.Transfections of HAE and 3T3 cells were performed in 96 well platedcontaining 20,000 cells plated 24 h earlier. In the transfectioncomplex, peptide to DNA charge ratios (±) were used at 1.5:1, 3:1 and7:1. The lipid component was maintained at a constant proportion, byweight, relative to DNA of 0.75:1. Prior to making transfectioncomplexes the lipid component was diluted to a concentration of 15 μgper ml, the peptide was prepared at 0.1 mg/ml and the DNA was at 20 μgper ml. All dilutions were performed with OptiMEM reduced serum tissueculture medium (Life Technologies). Transfection complexes were made bymixing of components in the order 1) lipid then 2) peptide and finally3) DNA, then diluted with OptiMEM to a concentration relative to the DNAcomponent of 0.25 μg DNA per 200 μl which volume was added to each well.Each group was performed in replicates of six. The vector complexsuspension was then applied to cells within 5 minutes of preparation.Transfection incubations were performed at 37° C. for 4 h. Luciferasereporter gene assays in cell free extracts were performed after 48 hincubation using a kit from Promega according to the manufacturer'sprotocol. Light units were standardised to the protein concentrationwithin each extract. The results of the transfection experiments areshown in FIG. 3.

[0181] At a 7:1 charge ratio the transfection efficiency of complexescontaining peptide P was five fold-higher than the next best peptide ,peptide 12 at a 3:1 ratio. Peptide P was more than 150-fold better thanPeptide S at the charge ratio of 7:1 indicating that the transfectionefficiency was receptor specific. Complexes containing peptide P werealmost nine-fold higher than K₁₆, again indicating receptor specificity.This result also suggests that peptide S is less than a tenth as good intransfection complexes as peptide K₁₆. This may be explained by sterichindrance by the scrambled motif in peptide S.

[0182] The difference in transfection performance between peptides P andQ was unexpected as peptide Q(LQHKSMP) varies from P by a single aminoacid residue. This result suggests that binding properties alone are notsufficient to explain the transfection potential of the peptides. Theseresults also suggest that the LID vector complex system may beretargeted to other specific peptides described herein this report andmay be useful for targeted gene delivery to epithelial cells in vivo orin vitro.

Example 2

[0183] Example 2 is a similar series of experiment to Example 1, withrelatively minor changes in a number of conditions.

[0184] Cell Lines

[0185] The human airway epithelial cell line (HAEo-) was maintained inEagle's minimal essential medium (MEM) HEPES modification (Sigma, Poole)containing 10% foetal calf serum (FCS), penicillin and streptomycin, andL-glutamine. The mouse fibroblast cell line 3T3 and the humanneuroblastoma cell line IMR32 were grown in Dulbecco's MEM withGlutamax-1, without sodium pyruvate, with 4500 mg/L glucose, withpyridoxine (Gibco BRL) with 10% FCS, penicillin and streptomycin added.Neuro-2A cells were maintained in Dulbecco's MEM with Glutamax-l (GibcoBRL) with 10% FCS, sodium pyruvate, penicillin and streptomycin andnon-essential amino acids.

[0186] Panning Cells in Monolayer

[0187] HAEO-cells were grown to confluence in 24 well plates. Cells werewashed twice in TBS before blocking cells with 2 mls 2% Marvel, 5%BSA-TBS per well for 30 mins at 4° C. The blocker was removed and 2×10¹¹phage were added in. 1 ml of 2% Marvel, 5% BSA -TBS. The phage wereallowed to bind for 2 hours shaking at 4° C. before washing five timeswith 2% BSA-TBS for 5 mins shaking at 4° C., followed by another fivewashes with 2% BSA-TBS for a few seconds only. Phage were eluted by theaddition of 400□1 76 mM citrate buffer pH 2.5 to the wells for 10 minsshaking at 4° C. The eluate was removed and the remaining cells werelysed with 1 ml 30 mM Tris pH 8.0, 1 mM EDTA for 1 hour on ice. Thecells were scraped from the plate, the eluate transferred to aneppendorf, and vortexed briefly. This eluate was saved as thecell-associated fraction. The phage from this elution were titrated asplaque forming units (PFU) as described in the literature supplied withthe library by NEB, before amplification of the phage in E.coli ER2738cells as described in the literature. For the second round of panning,2×10¹¹ of the amplified phage from the previous round was used as theinput phage. However, in order to reduce the number of plastic andblocking molecule-binding phage isolated, four pre-selection steps ofadding the phage to a blocked well with no cells for 30 mins at 4° C.was carried out before adding the phage to the HAEO-cells. Thestringency of washing as also increased in both the second and thirdrounds by the addition of a 10 min wash at 4° C. using 1 ml 76 mMcitrate buffer pH3.5. For the third round, 2×10¹¹ amplified phage fromthe second round was preselected in 5 blocked wells containing no cellsfor 30 mins each, followed by 1 well for 1 hour at 4° C. Phage bindingand elution was as described for the second round. Following titrationof the third round eluate, single well isolated plaques were picked,amplified and purified for sequencing and clone binding characterisationby whole cell ELISA.

[0188] Phage Sequencing

[0189] The phage were purified from small scale PEG preps (see suppliersmethods) and single stranded phage DNA was prepared for sequencing usingthe method described in Phage display of Peptides and Proteins Edited byBrian K. Kay, Jill Winter and John McCafferty. Briefly, the protein coatwas removed from the sample by phenol chloroform extraction, and the DNApelleted by ethanol precipitation. Trace salt was washed from the pelletwith ice cold 70% ethanol before resuspending the DNA in TE.

[0190] Between 50 and 100 ng purified DNA was used in a Big Dyeterminator cycle sequencing reaction (ABI) using the −96 primer(5′-CCCTCATTAGCGTAACG-3′) supplied with the library and purified forloading by ethanol precipitation as described in Big Dye kitinstructions. The samples were run on an ABI 377 sequencer and theresults analysed using the Vector NTI program.

[0191] Whole Cell ELISA

[0192] Approx. 8×10⁴ HAE cells in 100 ml HBSS were added to each well ofa 96 well plate and incubated at 37° C. until cells had adhered. Thecells were washed gently in HBSS before blocking by the addition of 0.5%BSA in HBSS for 30 mins. 1×10¹⁰ phage particles in blocker were added toeach well and allowed to bind at room temperature for 40 mins. Unboundphage were removed by washing twice with HBSS, and bound phage werefixed to the cells by incubation in 3.7 % paraformaldehyde for 10 mins.Cells were washed in PBS and incubated in blocking buffer for 45 mins,followed by three washes in PBS. Bound phage were detected by theaddition of HRP-conjugated anti-M13 antibody diluted 1:5000 in blockingbuffer for 1 hour, before washing three times in PBS, developing theELISA with ABTS solution, and reading the absorbance at 405 nm.

[0193] Peptide Synthesis

[0194] The [K]16—forms of the cyclised peptides (as shown in Table 6)were synthesised by standard solid phase synthesis by Alta Biosciences,Birmingham, and the Department of Chemistry, UCL. TABLE 6 Phage SEQ.Peptide peptide ID. name Peptide synthesised SEQ. ID. LPHKSMP 5 P[K]₁₆-GACLPHKSMPCG 13 LQHKSMP 4 Q [K]₁₆-GACLQHKSMPCG 12 YGLPHLF 19 Y[K]₁₆-GACYGLPHLFCG 44 SERSMNF 7 E [K]₁₆-GACSERSMNFCG 27 VKSMVTH 6 V[K]₁₆-GACVKSMVTHCG 28 PSGAARA 3 G [K]₁₆-GACPSGAARACG 29 YKHPGFL 21 S/YS[K]₁₆-GACYKHPGFLCG 30 NSFMESR 22 ES [K]₁₆-GACNSFMESRCG 31 AGSARPA 23 GS[K]₁₆-GACAGSARPACG 32 PLSHQMK 24 QS [K]₁₆-GACPLSHQMKCG 33 HPPMSKL 25 PS[K]₁₆-GACHPPMSKLCG 34 RRETEWA 26 6 [K]₁₆-GACRRETEWACG 35

[0195] For the avoidance of doubt, all of the sequences in Table 5 formpart of the present invention.

[0196] Transfections

[0197] Lipopolyplex Formation

[0198] Complexes were allowed to form electrostatically in a tube byadding the following components in the following order.50 μl ofLipofectin (Life Technologies Ltd) diluted to a concentration of 30μg/ml in OptiMEM, followed by 70 μl peptide (at varying concentrationsin OptiMEM for optimisation of the peptide:DNA charge ratio in thecomplex), with 50 μl of the luciferase reporter plasmid pCILuc at aconcentration of 40 ug/ml in Optimem added finally. The complex wasmixed by pipetting briefly before diluting in Optimem to a final volumeof 1.57 mls.

[0199] Transfection

[0200] The media was removed from subconfluent HAEO-cells plated at2×10⁴ cells/well overnight in 96 well plates and 200 μl of complex(approx. 0.25 kg of plasmid DNA) added to each well, leaving minimaltime between preparing the complex and adding to the cells. Alltransfections were carried out in 6 wells each. The cells were incubatedwith the complexes for 4 hours before replacing with normal media for 48hours, after which reporter gene expression was analysed by luciferaseassay (Promega).

[0201] Luciferase Assay

[0202] The cells were rinsed twice with PBS before the addition of 100μl of reporter lysis buffer (Promega, diluted 1 in 5 in dH2O) to thecells for 20 mins at 4° C. before freeze-thawing. 20 μl of the lysatewas transferred to a white plate and the luciferase was measured by aLucyl luminometer following the addition of 100 μl of reagent.

[0203] The protein present in each transfection well was calculatedusing the Bio-Rad protein assay reagent (based on the Bradford assay),adding 20 μl from the luciferase test to 200 μl of the reagent diluted 1in 5, incubating for 10 mins at room temperature and reading theabsorbance at 590 nm. The total protein present per well was calculatedfrom comparison with a range of BSA standards.

[0204] The results of the transfection experiments are shown in FIG. 4.Transfection of HAEO-cells with phage derived peptides and theirscrambled controls was carried out with a range of peptide:DNA chargeratios including 1.5:1, 3:1 and 7:1. The ratio giving the highesttransfection efficiency (determined as RLU/mg) for each peptide is shownin the figure. Controls include cells with no transfection complexesadded (OptiMEM only) and peptide 6, an integrin binding peptide. Eachresult is the mean of 6 values and error bars represent the standarddeviation about the mean.

Example 3

[0205] The transfection experiments described above were repeated usingNeuro-2A cells, IMR32 cells, rabbit adventitial fibroblast cells and 3T3cells. For analysis of transfections of those cell lines, cells wereplated to subconfluence overnight before transfecting in the same manneras above, analysing reporter gene expression after 24 hours. The resultsare shown in FIGS. 5 to 8.

[0206] Transfection of Neuro-2A cells with phage-derived peptides wascarried out with a range of peptide:DNA charge ratios including 1.5:1,3:1 and 7:1. The ratio giving the highest transfection efficiency(determined as RLU/mg) for each peptide is shown in FIG. 5. Controlsincluded cells with no transfection complexes added (OptiMEM only)peptide 6, an integrin binding peptide, and peptide S, the scrambledversion of peptide Y. Each result is the mean of 6 values and error barsrepresent the standard deviation about the mean.

[0207] Transfection of IMR32 cells with phage-derived peptides wascarried out with a range of peptide:DNA charge ratios including 1.5:1,3:1 and 7:1. The ratio giving the highest transfection efficiency(determined as RLU/mg) for each peptide is shown in FIG. 6. Controlsinclude cells with no transfection complexes added (OptiMEM only)peptide 6, an integrin binding peptide, and peptide S, the scrambledversion of peptide Y. Each result is the mean of 6 values and error barsrepresent the standard deviation about the mean.

[0208] Transfection of rabbit adventitial fibroblast cells withphage-derived peptides was carried out with a range of peptide:DNAcharge ratios including 1.5:1, 3:1 and 7:1. The ratio giving the highesttransfection efficiency (determined as RLU/mg)for each peptide is shownFIG. 7. Controls include cells with no transfection complexes added(OptiMEM only) peptide 6, an integrin binding peptide, and peptide S,the scrambled version of peptide Y. Each result is the mean of 6 valuesand error bars represent the standard deviation about the mean.

[0209] Transfection of 3T3 cells with phage-derived peptides was carriedout with a range of peptide:DNA charge ratios including 1.5:1, 3:1 and7:1. The ratio giving the highest transfection efficiency (determined asRLU/mg) for each peptide is shown in FIG. 8. Controls include cells withno transfection complexes added (OptiMEM only) peptide 6, an integrinbinding peptide, and peptide S, the scrambled version of peptide Y. Eachresult is the mean of 6 values and error bars represent the standarddeviation about the mean.

[0210] It is seen in FIGS. 5, 6 and 7 that transfection of Neuro-2Acells, IMR32 cells, rabbit adventitial fibroblast cells with thepeptides was of similar efficiency or lower than transfection withpeptide 6. Only in 3T3 cells (FIG. 8) was the transfection efficiencyabove that seen with peptide 6, with peptides Q and E showingefficiencies of approximately 1.5 times that seen with peptide 6. Alltransfections showed efficiencies above that of the scrambled peptide.These results may suggest that the molecules bound by the peptide arepresent on other cell types and in other species but maybe in alteredforms or at different densities compared to HAEO-cells.

[0211] References

[0212] 1. Wu G Y, Wu C H. Receptor-mediated in vitro gene transformationby a soluble DNA carrier system. J Biological Chemistry1987;262(10):4429-4432.

[0213] 2. Wagner E, Cotten M, Mechtler K, Kirlappos H, Birnstiel ML.DNA-binding transferrin conjugates as functional gene-delivery agents:Synthesis by linkage of polylysine or ethidium bromide to thetransferrin carbohydrate moiety. Bioconjugate Chemistry 1991;2:226-231.

[0214] 3. Cotten M, Lange. Transferrin-polycation-mediated introductionof DNA into human leukemic cells: Stimulation by agents that affect thesurvival of transfected DNA or modulate transferrin receptor levels.PNAS 1990;87:4033-4037.

[0215] 4. Ferkol T, Perales J C, Eckman E, Kaetzel C S, Hanson R W,Davis P B. Gene transfer into the airway epithelium of animals bytargeting the polymeric immunoglobulin receptor. J ClinicalInvestigation 1995;95:493-502.

[0216] 5. Curiel D T, Agarwal S, Wagner E, Cotten M. Adenovirusenhancement of transferrin-polylysine-mediated gene delivery. PNAS1991;88:8850-8854.

[0217] 6. Fernandez M A, Muno-Fernandez M A, Fresno M. Involvement of β1integrins in the binding and entry of Trypanosoma cruzi into humanmacrophages. European J of Immunology 1993;23:552-557.

[0218] 7. Wickham T J, Filardo E J, Cheresh D A, Nemerow G R. Integrin_(α)v-β5 selectively promotes adenovirus mediated cell membranepermeabilization. J Cell Biology 1994;127(l):257-264.

[0219] 8. Bergelson J M, Shepley M P, Chan B M C, Hemler M E, Finberg RW. identification of the integrin VLA-2 as a receptor for echovirus 1.Science 1992;255:1718-1720.

[0220] 9. Logan D, Abu-Ghazaleh R, Blakemore W, et al. Structure of amajor immunogenic site on foot-and-mouth disease virus. Nature1993;362:566-568.

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[0222] 11. Almeida E A C, Huovilla A-P J, Sutherland A E, et al. Mouseegg integrin _(α)6β1 functions as a sperm receptor. Cell1995;81:1095-1104.

[0223] 12. Clements J M, Newham P, Shepherd M, et al. Identification ofa key integrin-binding sequence in VCAM-1 homologous to the LDV activesite in fibronectin. J Cell Science 1994;107:2127-2135.

[0224] 13. Lu X, Deadman J J, Williams J A, Kakkar W, Rahman S.Synthetic RGD peptides derived from the adhesive domains of snake-venomproteins: evaluation as inhibitors of platelet aggregation. BiochemistryJ 1993;296:21-24.

[0225] 14. Koivunen E, Wang B, Ruoslahti E. Phage libraries displayingcyclic peptides with different ring sizes: ligand specificities of theRGD-directed integrins. Biol/Technology 1995;13:265-270.

[0226] 15. Koivunen E, Gay D A, Ruoslahti E. Selection of peptidesbinding to the _(α)5β1 integrin from phage display library. J BiologicalChemistry 1993;268(27):20205-20210.

[0227] 16. Koivunen E, Wang B, Ruoslahti E. Isolation of a highlyspecific ligand for the _(α)5β1 integrin from a phage display library. JCell Biology 1994;124(3):373-380.

[0228] 17. O'Neil K T, Hoess R H, Jackson A, Ramachandran N S, Mousa A,DeGrado W F. Identification of novel peptide antagonists for GPIIb/IIIafrom a conformationally constrained phage peptide library. Proteins1992;14:509-515.

[0229] 18.Healy J M, Murayama O, Maeda T, Yoshino K, Sekiguchi K,Kikuchi M. Peptide ligands for integrin alpha v beta 3 selected fromrandom phage display libraries. Biochemistry 1995;34:3948-3955.

[0230] 19. Pasqualani R, Koivunen E, Ruoslahti E. A peptide isolatedfrom phage display libraries is a structural and functional mimic of anRGD-binding site on integrins. J Cell Biology 1995;130:1189-1196.

[0231] 20. Hart S L, Knight A M, Harbottle R P, et al. Cell binding andinternalization by filamentous phage displaying a cyclicArg-Gly-Asp-containing peptide. J Biological Chemistry1994;269:12468-12474.

[0232] 21. Hart S L, Harbottle R P, Cooper R, Miller A, Williamson R,Coutelle C. Gene delivery and expression mediated by an integrin-bindingpeptide. Gen Therapy 1995;2:552-554.

[0233] 22. Wolfert M A, Seymour L W. Atomic force microscopic analysisof the influence of the molecular weight of poly(L)lysine on the size ofpolyelectrolyte complexes formed with DNA. Gene Therapy 1996;3:269-273.

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[0235] 24. Farhood H, Sebina A, Huang L. The role of dioleylphos-phatidylethanolamine (DOPE) in cationic liposome mediated genetransfer. Biochem Biophys Acta 1995;1235:289-295.

[0236] 25. Anderson R, MacDonald I, Corbett T, Hacking G, Lowdell M Wand Prentice H G. Human Gene Therapy 1997;8:1125-1135.

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[0238] 27. Bettinger, T., Remy, J. S. and Erbacher, P. (1999) Sizereduction of galactosylated PEI/DNA complexes improves lectin-mediatedgene transfer into hepatocytes. Bioconjug Chem, 10, 558-61.

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[0240] 29. Feero, W. G., Li, S., Rosenblatt, J. D., Sirianni, N.,Morgan, J. E., Partridge, T. A., Huang, L. and Hoffman, E. P. (1997)Selection and use of ligands for receptor-mediated gene delivery tomyogenic cells. Gene Ther, 4, 664-74.

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[0245] 34. Reddy, J. A., Dean, D., Kennedy, M. D. and Low, P. S. (1999)Optimization of folate-conjugated liposomal vectors for folatereceptor-mediated gene therapy. J Pharm Sci, 88, 1112-8.

[0246] 35. Reddy, J. A. and Low, P. S. (2000) Enhanced folate receptormediated gene therapy using a novel pH-sensitive lipid formulation. JControlled Release, 64, 27-37.

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1 44 1 3 PRT Artificial Sequence Targeting peptide, see description 1Xaa Ser Met 1 2 4 PRT Artificial Sequence Targeting peptide, seedescription 2 Leu Xaa His Lys 1 3 7 PRT Artificial Sequence Targetingpeptide, see description 3 Pro Ser Gly Ala Ala Arg Ala 1 5 4 7 PRTArtificial Sequence Targeting peptide, see description 4 Leu Gln His LysSer Met Pro 1 5 5 7 PRT Artificial Sequence Targeting peptide, seedescription 5 Leu Pro His Lys Ser Met Pro 1 5 6 7 PRT ArtificialSequence Targeting peptide, see description 6 Val Lys Ser Met Val ThrHis 1 5 7 7 PRT Artificial Sequence Targeting peptide, see description 7Ser Glu Arg Ser Met Asn Phe 1 5 8 7 PRT Artificial Sequence Targetingpeptide, see description 8 Val Gly Leu Pro His Lys Phe 1 5 9 7 PRTArtificial Sequence Targeting peptide, see description 9 Pro Ser Gly XaaAla Arg Ala 1 5 10 9 PRT Artificial Sequence Targeting peptide, seedescription 10 Cys Leu Pro His Lys Ser Met Pro Cys 1 5 11 9 PRTArtificial Sequence Targeting peptide, see description 11 Cys Leu GlnHis Lys Ser Met Pro Cys 1 5 12 28 PRT Artificial Sequence Targetingpeptide, see description 12 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 1 5 10 15 Gly Ala Cys Leu Gln His Lys Ser Met ProCys Gly 20 25 13 28 PRT Artificial Sequence Targeting peptide, seedescription 13 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys 1 5 10 15 Gly Ala Cys Leu Pro His Lys Ser Met Pro Cys Gly 20 2514 28 PRT Artificial Sequence Targeting peptide, see description 14 LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15Gly Ala Cys Tyr Lys His Pro Gly Phe Leu Cys Gly 20 25 15 31 PRTArtificial Sequence Targeting peptide, see description 15 Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Xaa SerXaa Gly Ala Cys Arg Arg Glu Thr Ala Trp Ala Cys Gly 20 25 30 16 7 PRTArtificial Sequence Targeting peptide, see description 16 Ser Xaa ArgSer Met Asn Phe 1 5 17 7 PRT Artificial Sequence Targeting peptide, seedescription 17 Ser Xaa Arg Ser Met Asn Phe 1 5 18 7 PRT ArtificialSequence Targeting peptide, see description 18 Leu Xaa His Lys Ser MetPro 1 5 19 7 PRT Artificial Sequence Targeting peptide, see description19 Tyr Gly Leu Pro His Lys Phe 1 5 20 7 PRT Artificial SequenceTargeting peptide, see description 20 Ser Glu Arg Ser Met Asn Phe 1 5 217 PRT Artificial Sequence Targeting peptide, see description 21 Tyr LysHis Pro Gly Phe Leu 1 5 22 7 PRT Artificial Sequence Targeting peptide,see description 22 Asn Ser Phe Met Glu Ser Arg 1 5 23 7 PRT ArtificialSequence Targeting peptide, see description 23 Ala Gly Ser Ala Arg ProAla 1 5 24 7 PRT Artificial Sequence Targeting peptide, see description24 Pro Leu Ser His Gln Met Lys 1 5 25 7 PRT Artificial SequenceTargeting peptide, see description 25 His Pro Pro Met Ser Lys Leu 1 5 267 PRT Artificial Sequence Targeting peptide, see description 26 Arg ArgGlu Thr Glu Trp Ala 1 5 27 28 PRT Artificial Sequence Targeting peptide,see description 27 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys 1 5 10 15 Gly Ala Cys Ser Glu Arg Ser Met Asn Phe Cys Gly 2025 28 28 PRT Artificial Sequence Targeting peptide, see description 28Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 1015 Gly Ala Cys Val Lys Ser Met Val Thr His Cys Gly 20 25 29 28 PRTArtificial Sequence Targeting peptide, see description 29 Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly AlaCys Pro Ser Gly Ala Ala Arg Ala Cys Gly 20 25 30 28 PRT ArtificialSequence Targeting peptide, see description 30 Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly Ala Cys Tyr LysHis Pro Gly Phe Leu Cys Gly 20 25 31 28 PRT Artificial SequenceTargeting peptide, see description 31 Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly Ala Cys Asn Ser Phe MetGlu Ser Arg Cys Gly 20 25 32 28 PRT Artificial Sequence Targetingpeptide, see description 32 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 1 5 10 15 Gly Ala Cys Ala Gly Ser Ala Arg Pro AlaCys Gly 20 25 33 28 PRT Artificial Sequence Targeting peptide, seedescription 33 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys 1 5 10 15 Gly Ala Cys Pro Leu Ser His Gln Met Lys Cys Gly 20 2534 28 PRT Artificial Sequence Targeting peptide, see description 34 LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15Gly Ala Cys His Pro Pro Met Ser Lys Leu Cys Gly 20 25 35 28 PRTArtificial Sequence Targeting peptide, see description 35 Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly AlaCys Arg Arg Glu Thr Glu Trp Ala Cys Gly 20 25 36 7 PRT ArtificialSequence Targeting peptide, see description 36 Ser Gln Arg Ser Met AsnPhe 1 5 37 7 PRT Artificial Sequence Targeting peptide, see description37 Gln Pro Leu Arg His His Gln 1 5 38 7 PRT Artificial SequenceTargeting peptide, see description 38 Pro Ser Gly Thr Ala Arg Ala 1 5 397 PRT Artificial Sequence Targeting peptide, see description 39 Lys GlnArg Pro Ala Trp Leu 1 5 40 7 PRT Artificial Sequence Targeting peptide,see description 40 Ile Pro Met Asn Ala Pro Trp 1 5 41 7 PRT ArtificialSequence Targeting peptide, see description 41 Ser Leu Pro Phe Ala ArgAsn 1 5 42 7 PRT Artificial Sequence Targeting peptide, see description42 Gly Pro Ala Arg Ile Ser Phe 1 5 43 7 PRT Artificial SequenceTargeting peptide, see description 43 Met Gly Leu Pro Leu Arg Phe 1 5 4428 PRT Artificial Sequence Targeting peptide, see description 44 Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 GlyAla Cys Tyr Gly Leu Pro His Leu Phe Cys Gly 20 25

1. A peptide having consisting of or comprising an amino acid sequenceselected from a) X¹SM [SEQ.ID.NO.:1]; b) LX²HK [SEQ.ID.NO.:2]; c)PSGX³ARA [SEQ.ID.NO.:9]; d) SX⁴RSMNF [SEQ.ID.NO.:16]; and e) LX⁵HKSMP[SEQ.ID.NO.:18], in which X¹ is a basic amino acid residue, X² is Q orP, X³ is A or T, X⁴ is an acidic amino acid residue and X⁵ is P or Q. 2.A peptide as claimed in claim 1 consisting of or comprising an aminoacid sequence selected from a) X¹SM [SEQ.ID.NO.:1]; b) LX²HK[SEQ.ID.NO.:2]; and c) PSGAARA [SEQ.ID.NO.:3], in which X¹ is a basicamino acid residue and X² is Q or P.
 3. A peptide as claimed in claim 1wherein X¹ is K or R.
 4. A peptide as claimed in claim 1 wherein X² isP.
 5. A peptide as claimed in claim 1 wherein X³ is A.
 6. A peptide asclaimed in claim 1 wherein X⁴ is E or Q.
 7. A peptide as claimed inclaim 6 wherein X⁴ is E.
 8. A peptide as claimed in claim 1 wherein X⁵is P.
 9. A peptide as claimed in any one of claims 1 to 3 wherein thepeptide comprises the sequence LQHKSMP [SEQ.ID.NO.4].
 10. A peptide asclaimed in any one of claims 1 to 4 or 8 wherein the peptide comprisesthe sequence LPHKSMP [SEQ.ID.NO.5].
 11. A peptide as claimed in any oneof claims 1, 2 or 3 wherein the peptide comprises the sequence VKSMVTH[SEQ.ID.NO.6].
 12. A peptide as claimed in any one of claims 1, 2, 3, 6or 7 wherein the peptide comprises the sequence SERSMNF [SEQ.ID.NO.7].13. A peptide as claimed in any one of claims 1, 2 or 4 wherein thepeptide comprises the sequence VGLPHKF [SEQ.ID.NO.8].
 14. A peptide asclaimed in any one of claims 1, 2 or 4 wherein the peptide comprises thesequence YGLPHKF [SEQ.ID.NO.19].
 15. A peptide as claimed in any one ofclaims 1, 2 or 5 wherein the peptide comprises the sequence PSGAARA[SEQ.ID.NO.3].
 16. A peptide as claimed in claim 1 wherein the peptidecomprises the sequence SQRSMNF [SEQ.ID.NO.:36].
 17. A peptide as claimedin claim 1 wherein the peptide comprises the sequence PSGTARA[SEQ.ID.NO.:38].
 18. A peptide as claimed in any one of claims 1 to 17having 5 to 20 amino acids.
 19. A peptide as claimed in any one ofclaims 1 to 17 having 6 to 12 amino acids.
 20. A peptide as claimed inany one of claims 1 to 17 having 7 amino acids.
 21. A peptide as claimedin any one of claims 1 to 20 comprising a cyclic region of amino acids.22. A peptide as claimed in claim 21 wherein the peptide comprises twoor more cysteine residues capable of forming one or more disulphidebond(s).
 23. A peptide as claimed in any one of claims 1 to 22 whereinthe peptide is linked to a polycationic nucleic acid-binding component.24. A peptide as claimed in claim 23 wherein the polycationic nucleicacid-binding component is polyethylenimine.
 25. A peptide as claimed inclaim 24 wherein the peptide is linked to the polyethylenimine via adisulphide bond.
 26. A peptide as claimed in claim 23 wherein thepolycationic nucleic acid-binding component is an oligo-lysine moleculehaving from 5 to 25 lysine moieties.
 27. A peptide as claimed in any oneof claims 23 to 26 wherein the peptide is linked to the polycationicnucleic acid-binding component via a spacer element.
 28. A peptide asclaimed in claim 27 wherein the spacer element is GG or GA or is longerand/or more hydrophobic than the dipeptide spacers GG (glycine-glycine)and GA (glycine-alanine).
 29. A peptide as claimed in claim 27 or 28wherein the spacer element is of formula GA.
 30. A peptide derivative offormula A-B-C wherein A is a polycationic nucleic acid-bindingcomponent, B is a spacer element, and C is a peptide as claimed in anyone of claims 1 to
 22. 31. A non-viral transfection complex thatcomprises (i) a nucleic acid, (ii) a lipid component, (iii) apolycationic nucleic acid-binding component, and (iv) a cell surfacereceptor binding component, comprising a peptide as claimed in any oneof claims 1 to
 30. 32. A complex as claimed in claim 31, wherein thenucleic acid component is or relates to a gene that is the target forgene therapy, gene vaccination or anti-sense therapy.
 33. A complex asclaimed in claim 31 or 32, wherein transcriptional and/or translationalcontrol elements for the nucleic acid are provided and the nucleic acidis optionally packed in a phage or vector.
 34. A complex as claimed inany one of claims 31 to 33, wherein the nucleic acid component is DNA.35. A complex as claimed in any one of claims 31 to 34, wherein thenucleic acid component is RNA.
 36. A complex as claimed in any one ofclaims 31 to 35, wherein the nucleic acid-binding component has from 3to 100 cationic monomers.
 37. A complex as claimed in any one of claims31 to 36, wherein the polycationic nucleic acid-binding component is anoligolysine.
 38. A complex as claimed in claim 37, wherein theoligolysine has from 10 to 20, especially 16 lysine residues.
 39. Acomplex as claimed in any one of claims 31 to 36, wherein thepolycationic nucleic acid-binding component is polyethylenimine.
 40. Acomplex as claimed in any one of claims 31 to 39, wherein the lipidcomponent is or is capable of forming a cationic liposome.
 41. A complexas claimed in any one of claims 31 to 40, wherein the lipid component isor comprises one or more lipids selected from cationic lipids and lipidshaving membrane destabilising or fusogenic properties.
 42. A complex asclaimed in claim 41, wherein the lipid component is or comprises theneutral lipid dioleyl phosphatidyl-ethanolamine (DOPE) or a lipid havingsimilar membrane destabilising or fusogenic properties.
 43. A complex asclaimed in claim 41 or claim 42, wherein the lipid component is orcomprises the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) ora lipid having similar cationic properties.
 44. A complex as claimed inclaim 43, wherein the lipid component is or comprises a mixture of DOPEand DOTMA, especially an equimolar mixture thereof.
 45. A complex asclaimed in claim 44, which comprises an equimolar mixture of DOPE andDOTMA as the lipid component, a peptide as claimed in any one of claims1 to 14 as the cell surface receptor-binding component, and [K]₁₆ as thepolycationic nucleic acid-binding component.
 46. A complex as claimed inclaim 44 or claim 45, wherein the ratio lipid component:the cell surfacereceptor-binding component/polycationic nucleic acid-binding component:nucleic acid is 0.75:4:1 by weight or 0.5 nmol:1.25 nmol:0.25 nmol on amolar basis.
 47. A complex as claimed in any one of claims 41 to 44,wherein the lipid component is or comprises2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoridoacetate(DOSPA) or a lipid having similar properties to those of DOSPA.
 48. Acomplex as claimed in claim 47, wherein the lipid component is orcomprises a mixture of DOPE and DOSPA, especially a mixture of one partby weight DOPE to 3 parts by weight DOSPA.
 49. A complex as claimed inclaim 48, which comprises a mixture of DOPE and DOSPA as the lipidcomponent, a peptide as claimed in any one of claims 1 to 14 as the cellsurface receptor-binding component, and [K]₁₆ as the polycationicnucleic acid-binding component.
 50. A complex as claimed in claim 49,wherein the ratio lipid component:polycationic nucleic acid-bindingcomponent: nucleic acid is 12:4:1 by weight.
 51. A process for theproduction of a complex as claimed in any one of claims 31 to 50, whichcomprises admixing components (i), (ii), (iii) and (iv).
 52. A processas claimed in claim 51, wherein the components are admixed in thefollowing order: lipid component, cell surface receptor-bindingcomponent/polycationic nucleic acid-binding component, nucleic acid. 53.A complex as claimed in any one of claims 31 to 50, obtainable by aprocess as claimed in claim 51 or claim
 52. 54. A non-viral transfectioncomplex that comprises (i) a nucleic acid, (iii) a polycationic nucleicacid-binding component, and (iv) a cell surface receptor bindingcomponent, comprising a peptide as claimed in any one of claims 1 to 30.55. A complex as claimed in claim 54, wherein the nucleic acid componentis as described in any one of claims 32 to
 35. 56. A complex as claimedin claim 54 or 55, wherein the polycationic nucleic acid-bindingcomponent is polyethylenimine.
 57. A complex as claimed in any one ofclaims 54 to 56, wherein the lipid component is or is capable of forminga cationic liposome.
 58. A process for the production of a complex asclaimed in any one of claims 54 to 57, which comprises admixingcomponents (i), (iii) and (iv).
 59. A process as claimed in claim 58,wherein the components are admixed in the following order: cell surfacereceptor-binding component/polycationic nucleic acid-binding component,nucleic acid.
 60. A complex as claimed in any one of claims 54 to 57obtainable by a process as claimed in claim 58 or claim
 59. 61. Amixture comprising a cell surface receptor-binding component, apolycationic nucleic acid-binding component, and a lipid component, thecell surface receptor-binding component being a peptide as defined inclaim
 1. 62. A mixture as claimed in claim 61 wherein the cell surfacereceptor-binding component is a peptide as defined in any one of claims2 to
 22. 63. A mixture as claimed in claim 61 or claim 62, wherein thepolycationic nucleic acid-binding component is as defined in any one ofclaims 36 to
 39. 64. A mixture as claimed in any one of claims 61 to 63,wherein the lipid component is as defined in any one of claims 40 to 44,47 and
 48. 65. A mixture as claimed in claim 44 which comprises anequimolar mixture of DOPE and DOTMA as the lipid component, a peptide asclaimed in any one of claims 1 to 22 as the cell surfacereceptor-binding component, and [K]₁₆ as the polycationic componentnucleic acid-binding component.
 66. A mixture as claimed in claim 65,wherein the ratio lipid component:combined cell surfacereceptor-binding/polycationic nucleic acid-binding component is 0.75:4by weight.
 67. A mixture comprising a cell surface receptor-bindingcomponent and a polycationic nucleic acid-binding component, the cellsurface receptor-binding component being a peptide as defined inclaim
 1. 68. A mixture as claimed in claim 67 wherein the cell surfacereceptor-binding component is a peptide as defined in any one of claims2 to
 22. 69. A mixture as claimed in claim 67 or claim 68, wherein thepolycationic nucleic acid-binding component is as defined in any one ofclaims 36 to
 39. 70. A process for producing a complex as claimed inclaim 31, which comprises incorporating a nucleic acid with a mixture asclaimed in any one of claims 61 to
 66. 71. A process for producing acomplex as claimed in claim 54, which comprises incorporating a nucleicacid with a mixture as claimed in any one of claims 67 to
 69. 72. Amethod of transfecting a cell with a nucleic acid, which comprisescontacting the cell in vitro or in vivo with a complex as claimed in anyone of claims 31 to 50, 53 to 57 or claim
 60. 73. A pharmaceuticalcomposition which comprises a complex as claimed in any one of claims 31to 50, 53 to 57 or claim 60, in admixture or conjunction with apharmaceutically suitable carrier.
 74. A method for the treatment orprophylaxis of a condition caused in human or in a non-human animal by adefect and/or a deficiency in a gene, which comprises administering acomplex as claimed in any one of claims 31 to 50, 53 to 57 or claim 60to the human or to the non-human animal.
 75. A method for therapeutic orprophylactic immunisation of a human or of a non-human animal, whichcomprises administering a complex as claimed in any one of claims 31 to50, 53 to 57 or claim 60 to the human or to the non-human animal.
 76. Amethod of anti-sense therapy, which comprises administering a complex asclaimed in any one of claims 31 to 50, 53 to 57 or claim 60 to a humanor to a non-human animal.
 77. A complex as claimed in any one of claims31 to 50, 53 to 57 or claim 60 for use as a medicament or a vaccine. 78.Use of a complex as claimed in any one of claims 31 to 50, 53 to 57 orclaim 60 for the manufacture of a medicament for the prophylaxis of acondition caused in a human or a non-human animal by a defect and/or adeficiency in a gene, or for therapeutic or prophylactic immunisation,or for anti-sense therapy.
 79. A kit that comprises (i) nucleic acid,(ii) a lipid component, (iii) a polycationic nucleic acid-bindingcomponent, and (iv) a cell surface receptor binding component,comprising a peptide as claimed in any one of claims 1 to
 30. 80. A kitthat comprises (i) nucleic acid, (iii) a polycationic nucleicacid-binding component, and (iv) a cell surface receptor bindingcomponent, comprising a peptide as claimed in any one of claims 1 to 30.81. A method for identifying a cell surface receptor binding ligand foruse in a non-viral transfection vector complex comprising the steps: a)selecting phage from a phage peptide library according to their bindingaffinity to cells of interest by bringing the phage into contact withthe cells of interest and washing away non-binding phage and thenextracting bound phage particles, b) repeating step a) if necessary c)selecting from the phage obtained in steps a) and b) phage which bind tothe cell of interest with high affinity using a whole cell ELISA.